Plants and seeds of corn variety cv032539

ABSTRACT

According to the invention, there is provided seed and plants of the corn variety designated CV032539. The invention thus relates to the plants, seeds and tissue cultures of the variety CV032539, and to methods for producing a corn plant produced by crossing a corn plant of variety CV032539 with itself or with another corn plant, such as a plant of another variety. The invention further relates to corn seeds and plants produced by crossing plants of variety CV032539 with plants of another variety, such as another inbred line. The invention further relates to the inbred and hybrid genetic complements of plants of variety CV032539.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of corn breeding.In particular, the invention relates to corn seed and plants of thevariety designated CV032539, and derivatives and tissue culturesthereof.

2. Description of Related Art

The goal of field crop breeding is to combine various desirable traitsin a single variety/hybrid. Such desirable traits include greater yield,better stalks, better roots, resistance to insecticides, herbicides,pests, and disease, tolerance to heat and drought, reduced time to cropmaturity, better agronomic quality, higher nutritional value, anduniformity in germination times, stand establishment, growth rate,maturity, and fruit size.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant. A plant cross-pollinates if pollen comes to it from aflower on a different plant.

Corn plants (Zea mays L.) can be bred by both self-pollination andcross-pollination. Both types of pollination involve the corn plant'sflowers. Corn has separate male and female flowers on the same plant,located on the tassel and the ear, respectively. Natural pollinationoccurs in corn when wind blows pollen from the tassels to the silks thatprotrude from the tops of the ear shoot.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants produces a uniform population ofhybrid plants that are heterozygous for many gene loci. Conversely, across of two plants each heterozygous at a number of loci produces apopulation of hybrid plants that differ genetically and are not uniform.The resulting non-uniformity makes performance unpredictable.

The development of uniform corn plant hybrids requires the developmentof homozygous inbred plants, the crossing of these inbred plants, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionare examples of breeding methods used to develop inbred plants frombreeding populations. Those breeding methods combine the geneticbackgrounds from two or more inbred plants or various other broad-basedsources into breeding pools from which new inbred plants are developedby selfing and selection of desired phenotypes. The new inbreds arecrossed with other inbred plants and the hybrids from these crosses areevaluated to determine which of those have commercial potential.

North American farmers plant tens of millions of acres of corn at thepresent time and there are extensive national and internationalcommercial corn breeding programs. A continuing goal of these cornbreeding programs is to develop corn hybrids that are based on stableinbred plants and have one or more desirable characteristics. Toaccomplish this goal, the corn breeder must select and develop superiorinbred parental plants.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a corn plant of thevariety designated CV032539. Also provided are corn plants having all ofthe morphological and physiological characteristics of the inbred cornvariety CV032539. The inbred corn plant of the invention may furthercomprise, or have, a cytoplasmic or nuclear factor that is capable ofconferring male sterility or otherwise preventing self-pollination, suchas by self-incompatibility. Parts of the corn plant of the presentinvention are also provided, for example, pollen obtained from an inbredplant and an ovule of the inbred plant.

The invention also concerns seed of the inbred corn variety CV032539.The inbred corn seed of the invention may be provided as an essentiallyhomogeneous population of inbred corn seed of the variety designatedCV032539. Essentially homogeneous populations of inbred seed aregenerally free from substantial numbers of other seed. Therefore, in thepractice of the present invention, inbred seed generally forms at leastabout 97% of the total seed. The population of inbred corn seed of theinvention may be particularly defined as being essentially free fromhybrid seed. The inbred seed population may be separately grown toprovide an essentially homogeneous population of inbred corn plantsdesignated CV032539.

In a further aspect of the invention, a composition is providedcomprising a seed of corn variety CV032539 comprised in plant seedgrowth media. In certain embodiments, the plant seed growth media is asoil or synthetic cultivation medium. In specific embodiments, thegrowth medium may be comprised in a container or may, for example, besoil in a field.

In another aspect of the invention, a plant of corn variety CV032539comprising an added heritable trait is provided. The heritable trait maycomprise a genetic locus that is a dominant or recessive allele. In oneembodiment of the invention, a plant of corn variety CV032539 furthercomprising a single locus conversion in particular is provided. Inspecific embodiments of the invention, an added genetic locus confersone or more traits such as, for example, male sterility, herbicidetolerance, insect resistance, disease resistance, waxy starch, modifiedfatty acid metabolism, modified phytic acid metabolism, modifiedcarbohydrate metabolism and modified protein metabolism. The trait maybe, for example, conferred by a naturally occurring maize geneintroduced into the genome of the variety by backcrossing, a natural orinduced mutation, or a transgene introduced through genetictransformation techniques. When introduced through transformation, agenetic locus may comprise one or more transgenes integrated at a singlechromosomal location.

In yet another aspect of the invention, an inbred corn plant of thevariety designated CV032539 is provided, wherein acytoplasmically-inherited trait has been introduced into said inbredplant. Such cytoplasmically-inherited traits are passed to progenythrough the female parent in a particular cross. An exemplarycytoplasmically-inherited trait is the male sterility trait.Cytoplasmic-male sterility (CMS) is a pollen abortion phenomenondetermined by the interaction between the genes in the cytoplasm and thenucleus. Alteration in the mitochondrial genome and the lack of restorergenes in the nucleus will lead to pollen abortion. With either a normalcytoplasm or the presence of restorer gene(s) in the nucleus, the plantwill produce pollen normally. A CMS plant can be pollinated by amaintainer version of the same variety, which has a normal cytoplasm butlacks the restorer gene(s) in the nucleus, and continue to be malesterile in the next generation. The male fertility of a CMS plant can berestored by a restorer version of the same variety, which must have therestorer gene(s) in the nucleus. With the restorer gene(s) in thenucleus, the offspring of the male-sterile plant can produce normalpollen grains and propagate. A cytoplasmically inherited trait may be anaturally occurring maize trait or a trait introduced through genetictransformation techniques.

In another aspect of the invention, a tissue culture of regenerablecells of a plant of variety CV032539 is provided. The tissue culturewill preferably be capable of regenerating plants capable of expressingall of the morphological and physiological characteristics of thevariety, and of regenerating plants having substantially the samegenotype as other plants of the variety. Examples of some of themorphological and physiological characteristics that may be assessedinclude characteristics related to yield, maturity, and kernel quality.The regenerable cells in such tissue cultures will preferably be derivedfrom embryos, meristematic cells, immature tassels, microspores, pollen,leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs,husks, or stalks, or from callus or protoplasts derived from thosetissues. Still further, the present invention provides corn plantsregenerated from the tissue cultures of the invention, the plants havingall of the morphological and physiological characteristics of varietyCV032539.

In yet another aspect of the invention, processes are provided forproducing corn seeds or plants, which processes generally comprisecrossing a first parent corn plant as a male or female parent with asecond parent corn plant, wherein at least one of the first or secondparent corn plants is a plant of the variety designated CV032539. Theseprocesses may be further exemplified as processes for preparing hybridcorn seed or plants, wherein a first inbred corn plant is crossed with asecond corn plant of a different, distinct variety to provide a hybridthat has, as one of its parents, the inbred corn plant variety CV032539.In these processes, crossing will result in the production of seed. Theseed production occurs regardless of whether the seed is collected ornot.

In one embodiment of the invention, the first step in “crossing”comprises planting, preferably in pollinating proximity, seeds of afirst and second parent corn plant, and preferably, seeds of a firstinbred corn plant and a second, distinct inbred corn plant. Where theplants are not in pollinating proximity, pollination can nevertheless beaccomplished by transferring a pollen or tassel bag from one plant tothe other as described below.

A second step comprises cultivating or growing the seeds of said firstand second parent corn plants into plants that bear flowers (corn bearsboth male flowers (tassels) and female flowers (silks) in separateanatomical structures on the same plant). A third step comprisespreventing self-pollination of the plants, i.e., preventing the silks ofa plant from being fertilized by any plant of the same variety,including the same plant. This is preferably done by emasculating themale flowers of the first or second parent corn plant, (i.e., treatingor manipulating the flowers so as to prevent pollen production, in orderto produce an emasculated parent corn plant). Self-incompatibilitysystems may also be used in some hybrid crops for the same purpose.Self-incompatible plants still shed viable pollen and can pollinateplants of other varieties but are incapable of pollinating themselves orother plants of the same variety.

A fourth step may comprise allowing cross-pollination to occur betweenthe first and second parent corn plants. When the plants are not inpollinating proximity, this is done by placing a bag, usually paper orglassine, over the tassels of the first plant and another bag over thesilks of the incipient ear on the second plant. The bags are left inplace for at least 24 hours. Since pollen is viable for less than 24hours, this assures that the silks are not pollinated from other pollensources, that any stray pollen on the tassels of the first plant isdead, and that the only pollen transferred comes from the first plant.The pollen bag over the tassel of the first plant is then shakenvigorously to enhance release of pollen from the tassels, and the shootbag is removed from the silks of the incipient ear on the second plant.Finally, the pollen bag is removed from the tassel of the first plantand is placed over the silks of the incipient ear of the second plant,shaken again and left in place. Yet another step comprises harvestingthe seeds from at least one of the parent corn plants. The harvestedseed can be grown to produce a corn plant or hybrid corn plant.

The present invention also provides corn seed and plants produced by aprocess that comprises crossing a first parent corn plant with a secondparent corn plant, wherein at least one of the first or second parentcorn plants is a plant of the variety designated CV032539. In oneembodiment of the invention, corn seed and plants produced by theprocess are first generation (F₁) hybrid corn seed and plants producedby crossing an inbred in accordance with the invention with another,distinct inbred. The present invention further contemplates seed of anF₁ hybrid corn plant. Therefore, certain exemplary embodiments of theinvention provide an F₁ hybrid corn plant and seed thereof.

In still yet another aspect of the invention, the genetic complement ofthe corn plant variety designated CV032539 is provided. The phrase“genetic complement” is used to refer to the aggregate of nucleotidesequences, the expression of which sequences defines the phenotype of,in the present case, a corn plant, or a cell or tissue of that plant. Agenetic complement thus represents the genetic make up of an inbredcell, tissue or plant, and a hybrid genetic complement represents thegenetic make up of a hybrid cell, tissue or plant. The invention thusprovides corn plant cells that have a genetic complement in accordancewith the inbred corn plant cells disclosed herein, and plants, seeds anddiploid plants containing such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that variety CV032539 could be identified by any of themany well known techniques such as, for example, Simple Sequence LengthPolymorphisms (SSLPs) (Williams et al., Nucleic Acids Res.,18:6531-6535, 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., Science,280:1077-1082, 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by corn plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of an inbred corn plant of the invention with a haploidgenetic complement of a second corn plant, preferably, another, distinctinbred corn plant. In another aspect, the present invention provides acorn plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

In still yet another aspect, the present invention provides a method ofproducing an inbred corn plant derived from the corn variety CV032539,the method comprising the steps of: (a) preparing a progeny plantderived from corn variety CV032539, wherein said preparing comprisescrossing a plant of the corn variety CV032539 with a second corn plant;(b) crossing the progeny plant with itself or a second plant to producea seed of a progeny plant of a subsequent generation; (c) growing aprogeny plant of a subsequent generation from said seed of a progenyplant of a subsequent generation and crossing the progeny plant of asubsequent generation with itself or a second plant; and (d) repeatingthe steps for an additional 3-10 generations to produce an inbred cornplant derived from the corn variety CV032539. In the method, it may bedesirable to select particular plants resulting from step (c) forcontinued crossing according to steps (b) and (c). By selecting plantshaving one or more desirable traits, an inbred corn plant derived fromthe corn variety CV032539 is obtained which possesses some of thedesirable traits of corn variety CV032539 as well as potentially otherselected traits.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions of PlantCharacteristics

Barren Plants: Plants that are barren, i.e., lack an ear with grain, orhave an ear with only a few scattered kernels.

Cg: Colletotrichum graminicola rating. Rating times 10 is approximatelyequal to percent total plant infection.

CLN: Corn Lethal Necrosis (combination of Maize Chlorotic Mottle Virusand Maize Dwarf Mosaic virus) rating: numerical ratings are based on aseverity scale where 1=most resistant to 9=susceptible.

Cn: Corynebacterium nebraskense rating. Rating times 10 is approximatelyequal to percent total plant infection.

Cz: Cercospora zeae-maydis rating. Rating times 10 is approximatelyequal to percent total plant infection.

Dgg: Diatraea grandiosella girdling rating (values are percent plantsgirdled and stalk lodged).

Dropped Ears: Ears that have fallen from the plant to the ground.

Dsp: Diabrotica species root ratings (1=least affected to 9=severepruning).

Ear-Attitude: The attitude or position of the ear at harvest scored asupright, horizontal, or pendant.

Ear-Cob Color: The color of the cob, scored as white, pink, red, orbrown.

Ear-Cob Diameter: The average diameter of the cob measured at themidpoint.

Ear-Cob Strength: A measure of mechanical strength of the cobs tobreakage, scored as strong or weak.

Ear-Diameter: The average diameter of the ear at its midpoint.

Ear-Dry Husk Color: The color of the husks at harvest scored as buff,red, or purple.

Ear-Fresh Husk Color: The color of the husks 1 to 2 weeks afterpollination scored as green, red, or purple.

Ear-Husk Bract: The length of an average husk leaf scored as short,medium, or long.

Ear-Husk Cover: The average distance from the tip of the ear to the tipof the husks. Minimum value no less than zero.

Ear-Husk Opening: An evaluation of husk tightness at harvest scored astight, intermediate, or open.

Ear-Length: The average length of the ear.

Ear-Number Per Stalk: The average number of ears per plant.

Ear-Shank Internodes: The average number of internodes on the ear shank.

Ear-Shank Length: The average length of the ear shank.

Ear-Shelling Percent: The average of the shelled grain weight divided bythe sum of the shelled grain weight and cob weight for a single ear.

Ear-Silk Color: The color of the silk observed 2 to 3 days after silkemergence scored as green-yellow, yellow, pink, red, or purple.

Ear-Taper (Shape): The taper or shape of the ear scored as conical,semi-conical, or cylindrical.

Ear-Weight: The average weight of an ear.

Early Stand: The percent of plants that emerge from the ground asdetermined in the early spring.

ER: Ear rot rating (values approximate percent ear rotted).

Final Stand Count: The number of plants just prior to harvest.

GDUs: Growing degree units which are calculated by the Barger Method,where the heat units for a 24-h period are calculated as GDUs=[(Maximumdaily temperature+Minimum daily temperature)/2]−50. The highest maximumdaily temperature used is 86° F. and the lowest minimum temperature usedis 50° F.

GDUs to Shed: The number of growing degree units (GDUs) or heat unitsrequired for an inbred line or hybrid to have approximately 50% of theplants shedding pollen as measured from time of planting. GDUs to shedis determined by summing the individual GDU daily values from theplanting date to the date of 50% pollen shed.

GDUs to Silk: The number of growing degree units for an inbred line orhybrid to have approximately 50% of the plants with silk emergence asmeasured from the time of planting. GDUs to silk is determined bysumming the individual GDU daily values from the planting date to thedate of 50% silking.

Hc2: Helminthosporium carbonum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

Hc3: Helminthosporium carbonum race 3 rating. Rating times 10 isapproximately equal to percent total plant infection.

Hm: Helminthosporium maydis race 0 rating. Rating times 10 isapproximately equal to percent total plant infection.

Ht1: Helminthosporium turcicum race 1 rating. Rating times 10 isapproximately equal to percent total plant infection.

Ht2: Helminthosporium turcicum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

HtG: Chlorotic-lesion type resistance. +=indicates the presence of Htchlorotic-lesion type resistance; −=indicates absence of Htchlorotic-lesion type resistance; and +/−=indicates segregation of Htchlorotic-lesion type resistance. Rating times 10 is approximately equalto percent total plant infection.

Kernel-Aleurone Color: The color of the aleurone scored as white, pink,tan, brown, bronze, red, purple, pale purple, colorless, or variegated.

Kernel-Cap Color: The color of the kernel cap observed at dry stage,scored as white, lemon-yellow, yellow, or orange.

Kernel-Endosperm Color: The color of the endosperm scored as white, paleyellow, or yellow.

Kernel-Endosperm Type: The type of endosperm scored as normal, waxy, oropaque.

Kernel-Grade: The percent of kernels that are classified as rounds.

Kernel-Length: The average distance from the cap of the kernel to thepedicel.

Kernel-Number Per Row: The average number of kernels in a single row.

Kernel-Pericarp Color: The color of the pericarp scored as colorless,red-white crown, tan, bronze, brown, light red, cherry red, orvariegated.

Kernel-Row Direction: The direction of the kernel rows on the ear scoredas straight, slightly curved, spiral, or indistinct (scattered).

Kernel-Row Number: The average number of rows of kernels on a singleear.

Kernel-Side Color: The color of the kernel side observed at the drystage, scored as white, pale yellow, yellow, orange, red, or brown.

Kernel-Thickness: The distance across the narrow side of the kernel.

Kernel-Type: The type of kernel scored as dent, flint, or intermediate.

Kernel-Weight: The average weight of a predetermined number of kernels.

Kernel-Width: The distance across the flat side of the kernel.

Kz: Kabatiella zeae rating. Rating times 10 is approximately equal topercent total plant infection.

Leaf-Angle: Angle of the upper leaves to the stalk scored as upright (0to 30 degrees), intermediate (30 to 60 degrees), or lax (60 to 90degrees).

Leaf-Color: The color of the leaves 1 to 2 weeks after pollinationscored as light green, medium green, dark green, or very dark green.

Leaf-Length: The average length of the primary ear leaf.

Leaf-Longitudinal Creases: A rating of the number of longitudinalcreases on the leaf surface 1 to 2 weeks after pollination. Creases arescored as absent, few, or many.

Leaf-Marginal Waves: A rating of the waviness of the leaf margin 1 to 2weeks after pollination. Rated as none, few, or many.

Leaf-Number: The average number of leaves of a mature plant. Countingbegins with the cotyledonary leaf and ends with the flag leaf.

Leaf-Sheath Anthocyanin: A rating of the level of anthocyanin in theleaf sheath 1 to 2 weeks after pollination, scored as absent,basal-weak, basal-strong, weak or strong.

Leaf-Sheath Pubescence: A rating of the pubescence of the leaf sheath.Ratings are taken 1 to 2 weeks after pollination and scored as light,medium, or heavy.

Leaf-Width: The average width of the primary ear leaf measured at itswidest point.

LSS: Late season standability (values times 10 approximate percentplants lodged in disease evaluation plots).

Moisture: The moisture of the grain at harvest.

On1: Ostrinia nubilalis 1st brood rating (1=resistant to 9=susceptible).

On2: Ostrinia nubilalis 2nd brood rating (1=resistant to 9=susceptible).

Relative Maturity: A maturity rating based on regression analysis. Theregression analysis is developed by utilizing check hybrids and theirpreviously established day rating versus actual harvest moistures.Harvest moisture on the hybrid in question is determined and thatmoisture value is inserted into the regression equation to yield arelative maturity.

Root Lodging: Root lodging is the percentage of plants that root lodge.A plant is counted as root lodged if a portion of the plant leans fromthe vertical axis by approximately 30 degrees or more.

Seedling Color: Color of leaves at the 6 to 8 leaf stage.

Seedling Height: Plant height at the 6 to 8 leaf stage.

Seedling Vigor: A visual rating of the amount of vegetative growth on a1 to 9 scale, where 1 equals best. The score is taken when the averageentry in a trial is at the fifth leaf stage.

Selection Index: The selection index gives a single measure of hybrid'sworth based on information from multiple traits. One of the traits thatis almost always included is yield. Traits may be weighted according tothe level of importance assigned to them.

Sr: Sphacelotheca reiliana rating is actual percent infection.

Stalk-Anthocyanin: A rating of the amount of anthocyanin pigmentation inthe stalk. The stalk is rated 1 to 2 weeks after pollination as absent,basal-weak, basal-strong, weak, or strong.

Stalk-Brace Root Color: The color of the brace roots observed 1 to 2weeks after pollination as green, red, or purple.

Stalk-Diameter: The average diameter of the lowest visible internode ofthe stalk.

Stalk-Ear Height: The average height of the ear measured from the groundto the point of attachment of the ear shank of the top developed ear tothe stalk.

Stalk-Internode Direction: The direction of the stalk internode observedafter pollination as straight or zigzag.

Stalk-Internode Length: The average length of the internode above theprimary ear.

Stalk Lodging: The percentage of plants that did stalk lodge. Plants arecounted as stalk lodged if the plant is broken over or off below theear.

Stalk-Nodes With Brace Roots: The average number of nodes having braceroots per plant.

Stalk-Plant Height: The average height of the plant as measured from thesoil to the tip of the tassel.

Stalk-Tillers: The percent of plants that have tillers. A tiller isdefined as a secondary shoot that has developed as a tassel capable ofshedding pollen.

Staygreen: Staygreen is a measure of general plant health near the timeof black layer formation (physiological maturity). It is usuallyrecorded at the time the ear husks of most entries within a trial haveturned a mature color. Scoring is on a 1 to 9 basis where 1 equals best.

STR: Stalk rot rating (values represent severity rating of 1=25% ofinoculated internode rotted to 9=entire stalk rotted and collapsed).

SVC: Southeastern Virus Complex (combination of Maize Chlorotic DwarfVirus and Maize Dwarf Mosaic Virus) rating; numerical ratings are basedon a severity scale where 1=most resistant to 9=susceptible.

Tassel-Anther Color: The color of the anthers at 50% pollen shed scoredas green-yellow, yellow, pink, red, or purple.

Tassel-Attitude: The attitude of the tassel after pollination scored asopen or compact.

Tassel-Branch Angle: The angle of an average tassel branch to the mainstem of the tassel scored as upright (less than 30 degrees),intermediate (30 to 45 degrees), or lax (greater than 45 degrees).

Tassel-Branch Number: The average number of primary tassel branches.

Tassel-Glume Band: The closed anthocyanin band at the base of the glumescored as present or absent.

Tassel-Glume Color: The color of the glumes at 50% shed scored as green,red, or purple.

Tassel-Length: The length of the tassel measured from the base of thebottom tassel branch to the tassel tip.

Tassel-Peduncle Length: The average length of the tassel peduncle,measured from the base of the flag leaf to the base of the bottom tasselbranch.

Tassel-Pollen Shed: A visual rating of pollen shed determined by tappingthe tassel and observing the pollen flow of approximately five plantsper entry. Rated on a 1 to 9 scale where 9=sterile, 1=most pollen.

Tassel-Spike Length: The length of the spike measured from the base ofthe top tassel branch to the tassel tip.

Test Weight: Weight of the grain in pounds for a given volume (bushel)adjusted to 15.5% moisture.

Yield: Yield of grain at harvest adjusted to 15.5% moisture.

II. Other Definitions

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny back to one of the parents, for example, a first generationhybrid (F₁) with one of the parental genotypes of the F₁ hybrid.

Chromatography: A technique wherein a mixture of dissolved substancesare bound to a solid support followed by passing a column of fluidacross the solid support and varying the composition of the fluid. Thecomponents of the mixture are separated by selective elution.

Crossing: The pollination of a female flower of a corn plant, therebyresulting in the production of seed from the flower.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a chemical agent or a cytoplasmic or nuclear geneticfactor conferring male sterility.

F₁ Hybrid: The first generation progeny of the cross of two plants.

Genetic Complement: An aggregate of nucleotide sequences, the expressionof which sequences defines the phenotype in corn plants, or componentsof plants including cells or tissue.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Isozymes: Detectable variants of an enzyme, the variants catalyzing thesame reaction(s) but differing from each other, e.g., in primarystructure and/or electrophoretic mobility. The differences betweenisozymes are under single gene, codominant control. Consequently,electrophoretic separation to produce band patterns can be equated todifferent alleles at the DNA level. Structural differences that do notalter charge cannot be detected by this method.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Genetic loci that contribute, at least inpart, certain numerically representable traits that are usuallycontinuously distributed.

Regeneration: The development of a plant from tissue culture.

SSR profile: A profile of simple sequence repeats used as geneticmarkers and scored by gel electrophoresis following PCR™ amplificationusing flanking oligonucleotide primers.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing wherein essentially allof the morphological and physiological characteristics of an inbred arerecovered in addition to the characteristics conferred by the singlelocus transferred into the inbred via the backcrossing technique.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic sequence which has been introduced into the nuclearor chloroplast genome of a maize plant by a genetic transformationtechnique.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

III. Inbred Corn Plant CV032539

A. Origin and Breeding History

Inbred plant CV032539 was derived from a cross between the linesCV112211 and 1351729. The origin and breeding history of inbred plantCV032539 can be summarized as follows:

Winter 2004 The inbred line CV112211 (a proprietary Monsanto Companyinbred) was crossed to the inbred line I351729 (a proprietary MonsantoCompany inbred) in Kuanakakai, HI nursery 9A6-1, and F₁ seed producedand bulked. The F₁ seed was located in Range 00018:Column 00001. Summer2005 The F₁ seed produced in Winter 2004 was grown and self-pollinatedin Arlington, WI nursery field WIAR05F1 Range 00066:Column 00004 toproduce F₂ seed. The resulting F₂ seed was bulked. Summer 2006 The F₂seed produced in Summer 2005 was grown and self- pollinated in Glyndon,MN nursery MNGLSEGF1F2 rows 351-400. Fifty-eight F₃ ears were harvested.F₃ ear 0012 located in Range 00208:Column 00050 was selected foradvancement. Summer 2007 F₃ ear 0012 produced in Summer 2006 was plantedear-to-row in Glyndon nursery MNGLSEG and self pollinated. F₄ ear 0001located in Range 00114:Column 00020 was selected for advancement. The F₄generation was coded as CV032539. .Summer 2008 F₄ ear selection 0001produced in Summer 2007 (coded as CV032539) was planted ear-to-row inGlyndon, MN nursery field MNGLNUR1 and self-pollinated. Seed from F₄ earselection 0001 was also placed on two testers in summer ICBs at Mason,MI and Olivia, MN. F₅ ear 0002 located in Range 00113:Column 00076 wasselected for advancement. Winter 2008 F₅ ear selection 0002 produced inSummer 2008 was planted ear-to- row and self-pollinated in PuertoVallarta nursery field JAPVL1-SS- 1. Seed from F₅ ear selection 0002 wasalso placed on one tester in a winter ICB at Kunia, HI. F₆ ear 0001located in Range 00030:Column 00059 was selected for advancement. The F₆generation was finished as inbred line CV032539. Summer 2009 F₆ earselection 0001 produced in Winter 2008 was planted ear-to- row andself-pollinated in Glyndon, MN nursery field MNGLSEG1. F₇ ear 0002located in Range 00132:Column 00101 was selected for advancement andused to begin the breeder seed increase. Screening 2 hybrids producedduring Summer and Winter 2008 were tested.

Corn variety CV032539 shows uniformity and stability within the limitsof environmental influence for the traits described hereinafter inTable 1. CV032539 has been self-pollinated and ear-rowed a sufficientnumber of generations with careful attention paid to uniformity of planttype to ensure homozygosity and phenotypic stability. No variant traitshave been observed or are expected in CV032539.

Inbred corn plants can be reproduced by planting the seeds of the inbredcorn plant CV032539, growing the resulting corn plants underself-pollinating or sib-pollinating conditions with adequate isolationusing standard techniques well known to an artisan skilled in theagricultural arts. Seeds can be harvested from such a plant usingstandard, well known procedures.

B. Phenotypic Description

In accordance with another aspect of the present invention, there isprovided a corn plant having the morphological characteristics of cornplant CV032539. A description of the morphological and physiologicalcharacteristics of corn plant CV032539 is presented in Table 1.

TABLE 1 Morphological and Physiological Traits for Corn Variety CV032539VALUE CHARACTERISTIC CV032539 CV112211 I351729 1. STALK Plant Height(cm.) 174.4 209.1 179.4 Ear Height (cm) 65.3 67.9 59.7 Anthocyanin BasalWeak Absent Basal Weak Brace Root Color Moderate Faint ModerateInternode Direction Straight Straight Straight Internode Length cm. 15.715.4 15.7 2. LEAF Color Dark Green Green Dark Green Length cm. 71.9 70.667.1 Width cm. 7.9 9.9 7.9 Sheath Anthocyanin Absent Absent AbsentSheath Pubescence Very Light Very Light Very Light Marginal Waves FewFew Moderate Longitudinal Creases Many Many Moderate 3. TASSEL Lengthcm. 41.7 36.6 35.5 Peduncle Length cm. 11.3 5.5 5.6 Branch Number 7.94.5 9.5 Anther Color Yellow Salmon Yellow Glume Color Purple Green GreenGlume Band Absent Absent Absent 4. EAR Silk Color Green-Yellow YellowYellow Number Per Stalk 1.0 2.0 1.0 Position (attitude) Upright UprightUpright Length cm. 11.5 15.2 11.7 Shape Semi-Conical Semi- Semi- ConicalConical Diameter cm. 4.1 4.4 4.3 Shank Length cm. 17.5 12.6 13.2 HuskBract Short Short Short Husk Cover cm. 8.8 5.7 5.1 Husk Opening VeryTight Tight Tight Husk Color Fresh Green Green Green Husk Color Dry BuffBuff Buff Cob Diameter cm. 2.6 2.6 2.6 Cob Color Red Red White ShellingPercent 82.0 85.2 84.6 5. KERNEL Row Number 16.3 16.2 15.6 Number PerRow 16.8 31.4 19.2 Row Direction Slightly Curved Straight Straight TypeIntermediate Dent Dent Cap Color Yellow Deep Yellow Deep Yellow SideColor Deep Yellow Yellow- Orange Orange Length (depth) mm. 9.4 11.2 11.5Width mm. 7.2 7.9 7.9 Thickness 5.9 4.7 4.5 Endosperm Type Normal NormalNormal Endosperm Color Yellow Yellow Orange *These are typical values.Values may vary due to environment. Other values that are substantiallyequivalent are also within the scope of the invention.

C. Deposit Information

A deposit was made of at least 2500 seeds of corn variety CV032539 withthe American Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209 USA. The deposit was assigned ATCC AccessionNo. PTA-______. The date of deposit of the seeds with the ATCC was______. Access to this deposit will be available during the pendency ofthe application to the Commissioner of Patents and Trademarks andpersons determined by the Commissioner to be entitled thereto uponrequest. The deposit will be maintained in the ATCC Depository, which isa public depository, for a period of 30 years, or 5 years after the mostrecent request, or for the enforceable life of the patent, whichever islonger, and will be replaced if it becomes nonviable during that period.Applicant does not waive any infringement of their rights granted underthis patent or under the Plant Variety Protection Act (7 U.S.C. 2321 etseq.).

IV. Further Embodiments of the Invention

In one embodiment, compositions are provided comprising a seed of cornvariety CV032539 comprised in plant seed growth media. Plant seed growthmedia are well known to those of skill in the art and include, but arein no way limited to, soil or synthetic cultivation medium.Advantageously, plant seed growth media can provide adequate physicalsupport for seeds and can retain moisture and/or nutritional components.Examples of characteristics for soils that may be desirable in certainembodiments can be found, for instance, in U.S. Pat. Nos. 3,932,166 and4,707,176. Synthetic plant cultivation media are also well known in theart and may, in certain embodiments, comprise polymers or hydrogels.Examples of such compositions are described, for example, in U.S. Pat.No. 4,241,537.

In certain further aspects, the invention provides plants modified toinclude at least a first desired heritable trait. Such plants may, inone embodiment, be developed by a plant breeding technique calledbackcrossing, wherein essentially all of the morphological andphysiological characteristics of a variety are recovered in addition toa genetic locus transferred into the plant via the backcrossingtechnique. By essentially all of the morphological and physiologicalcharacteristics, it is meant that the characteristics of a plant arerecovered that are otherwise present when compared in the sameenvironment, other than an occasional variant trait that might ariseduring backcrossing or direct introduction of a transgene. It isunderstood that a locus introduced by backcrossing may or may not betransgenic in origin, and thus the term backcrossing specificallyincludes backcrossing to introduce loci that were created by genetictransformation.

Backcrossing methods can be used with the present invention to improveor introduce a trait into a variety. The term backcros sing as usedherein refers to the repeated crossing of a hybrid progeny back to oneof the parental corn plants. The parental corn plant which contributesthe locus or loci for the desired trait is termed the nonrecurrent ordonor parent. This terminology refers to the fact that the nonrecurrentparent is used one time in the backcross protocol and therefore does notrecur.

The parental corn plant to which the locus or loci from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman et al., In:Breeding Field Crops, 4^(th) Ed., Iowa State University Press, Ames,Iowa, 132-155; 321-344, 1995; Fehr, Principles of Cultivar Development,1:360-376, 1987; Sprague and Dudley, In: Corn and Corn Improvement,3^(rd) Ed., Crop Science of America, Inc.; Soil Science of America,Inc., Wisconsin. 881-883; 901-918, 1988). In a typical backcrossprotocol, the original parent of interest (recurrent parent) is crossedto a second variety (nonrecurrent parent) that carries the genetic locusof interest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a corn plant is obtained wherein essentially all of themorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the transferredlocus from the nonrecurrent parent. The backcross process may beaccelerated by the use of genetic markers, such as SSR, RFLP, SNP orAFLP markers to identify plants with the greatest genetic complementfrom the recurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto add or substitute one or more new traits in the original variety andprogeny therefrom. To accomplish this, a genetic locus of the recurrentparent is modified or substituted with the desired locus from thenonrecurrent parent, while retaining essentially all of the rest of thegenetic, and therefore the morphological and physiological constitutionof the original plant. The choice of the particular nonrecurrent parentwill depend on the purpose of the backcross; one of the major purposesis to add some commercially desirable, agronomically important trait tothe plant. The exact backcrossing protocol will depend on thecharacteristic or trait being altered to determine an appropriatetesting protocol. Although backcrossing methods are simplified when thecharacteristic being transferred is a dominant allele, a recessiveallele may also be transferred. In this instance it may be necessary tointroduce a test of the progeny to determine if the desiredcharacteristic has been successfully transferred.

Many traits have been identified that are not regularly selected for inthe development of a new variety but that can be improved bybackcrossing techniques. A genetic locus conferring the traits may ormay not be transgenic. Examples of such traits known to those of skillin the art include, but are not limited to, male sterility, waxy starch,herbicide resistance, resistance for bacterial, fungal, or viraldisease, insect resistance, male fertility and enhanced nutritionalquality. These genes are generally inherited through the nucleus, butmay be inherited through the cytoplasm. Some known exceptions to thisare genes for male sterility, some of which are inheritedcytoplasmically, but still act as a single locus trait.

Direct selection may be applied where a genetic locus acts as a dominanttrait. An example of a dominant trait is the herbicide resistance trait.For this selection process, the progeny of the initial cross are sprayedwith the herbicide prior to the backcrossing. The spraying eliminatesany plants which do not have the desired herbicide resistancecharacteristic, and only those plants which have the herbicideresistance gene are used in the subsequent backcross. This process isthen repeated for all additional backcross generations.

Many useful traits are those which are introduced by genetictransformation techniques. Methods for the genetic transformation ofcorn are known to those of skill in the art. For example, methods whichhave been described for the genetic transformation of corn includeelectroporation (U.S. Pat. No. 5,384,253), electrotransformation (U.S.Pat. No. 5,371,003), microprojectile bombardment (U.S. Pat. No.5,550,318; U.S. Pat. No. 5,736,369, U.S. Pat. No. 5,538,880; and PCTPublication WO 95/06128), Agrobacterium-mediated transformation (U.S.Pat. No. 5,591,616 and E.P. Publication EP672752), direct DNA uptaketransformation of protoplasts (Omirulleh et al., Plant Mol. Biol.,21(3):415-428, 1993) and silicon carbide fiber-mediated transformation(U.S. Pat. No. 5,302,532 and U.S. Pat. No. 5,464,765).

It is understood to those of skill in the art that a transgene need notbe directly transformed into a plant, as techniques for the productionof stably transformed corn plants that pass single loci to progeny byMendelian inheritance are well known in the art. Such loci may thereforebe passed from parent plant to progeny plants by standard plant breedingtechniques that are well known in the art. Non-limiting examples oftraits that may be introduced into a corn plant according to specificembodiments of the invention are provided below.

A. Male Sterility

Examples of genes conferring male sterility include those disclosed inU.S. Pat. No. 3,861,709, U.S. Pat. No. 3,710,511, U.S. Pat. No.4,654,465, U.S. Pat. No. 5,625,132, and U.S. Pat. No. 4,727,219, each ofthe disclosures of which are specifically incorporated herein byreference in their entirety. The use of herbicide-inducible malesterility genes is described in U.S. Pat. No. 6,762,344. Male sterilitygenes can increase the efficiency with which hybrids are made, in thatthey eliminate the need to physically emasculate the corn plant used asa female in a given cross.

Where one desires to employ male-sterility systems with a corn plant inaccordance with the invention, it may be beneficial to also utilize oneor more male-fertility restorer genes. For example, where cytoplasmicmale sterility (CMS) is used, hybrid seed production requires threeinbred lines: (1) a cytoplasmically male-sterile line having a CMScytoplasm; (2) a fertile inbred with normal cytoplasm, which is isogenicwith the CMS line for nuclear genes (“maintainer line”); and (3) adistinct, fertile inbred with normal cytoplasm, carrying a fertilityrestoring gene (“restorer” line). The CMS line is propagated bypollination with the maintainer line, with all of the progeny being malesterile, as the CMS cytoplasm is derived from the female parent. Thesemale sterile plants can then be efficiently employed as the femaleparent in hybrid crosses with the restorer line, without the need forphysical emasculation of the male reproductive parts of the femaleparent.

The presence of a male-fertility restorer gene results in the productionof fully fertile F₁ hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Such hybrids areuseful where the vegetative tissue of the corn plant is utilized, e.g.,for silage, but in most cases, the seeds will be deemed the mostvaluable portion of the crop, so fertility of the hybrids in these cropsmust be restored. Therefore, one aspect of the current inventionconcerns plants of the corn variety CV032539 comprising a genetic locuscapable of restoring male fertility in an otherwise male-sterile plant.Examples of male-sterility genes and corresponding restorers which couldbe employed with the plants of the invention are well known to those ofskill in the art of plant breeding and are disclosed in, for instance,U.S. Pat. No. 5,530,191; U.S. Pat. No. 5,689,041; U.S. Pat. No.5,741,684; and U.S. Pat. No. 5,684,242, the disclosures of which areeach specifically incorporated herein by reference in their entirety.

B. Herbicide Resistance

Numerous herbicide resistance genes are known and may be employed withthe invention. An example is a gene conferring resistance to a herbicidethat inhibits the growing point or meristem, such as an imidazalinone ora sulfonylurea. Exemplary genes in this category code for ALS and AHASenzymes as described, for example, by Lee et al., EMBO J., 7:1241, 1988;Gleen et al., Plant Molec. Biology, 18:1185-1187, 1992; and Miki et al.,Theor. Appl. Genet., 80:449, 1990.

Resistance genes for glyphosate (resistance conferred by mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively), and hygromycin B phosphotransferase, and to otherphosphono compounds such as glufosinate (phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicus phosphinothricin-acetyltransferase (bar) genes) may also be used. See, for example, U.S. Pat.No. 4,940,835 to Shah et al., which discloses the nucleotide sequence ofa form of EPSPS which can confer glyphosate resistance. Examples ofspecific EPSPS transformation events conferring glyphosate resistanceare provided by U.S. Pat. No. 6,040,497.

A DNA molecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. A hygromycin Bphosphotransferase gene from E. coli which confers resistance toglyphosate in tobacco callus and plants is described in Penaloza-Vazquezet al., Plant Cell Reports, 14:482-487, 1995. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyltransferase gene isprovided in European patent application No. 0 242 246 to Leemans et al.DeGreef et al. (Biotechnology, 7:61, 1989), describe the production oftransgenic plants that express chimeric bar genes coding forphosphinothricin acetyl transferase activity. Exemplary genes conferringresistance to phenoxy propionic acids and cyclohexanediones, such assethoxydim and haloxyfop, are the Acct-S1, Accl-S2 and Acct-S3 genesdescribed by Marshall et al. (Theor. Appl. Genet., 83:4:35, 1992).

Genes conferring resistance to a herbicide that inhibits photosynthesisare also known, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibila et al. (Plant Cell, 3:169,1991), describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA moleculescontaining these genes are available under ATCC Accession Nos. 53435,67441, and 67442. Cloning and expression of DNA coding for a glutathioneS-transferase is described by Hayes et al. (Biochem. J., 285(Pt1):173-180, 1992). Protoporphyrinogen oxidase (PPO) is the target of thePPO-inhibitor class of herbicides; a PPO-inhibitor resistant PPO genewas recently identified in Amaranthus tuberculatus (Patzoldt et al.,PNAS, 103(33):12329-2334, 2006). The herbicide methyl viologen inhibitsCO₂ assimilation. Foyer et al. (Plant Physiol., 109:1047-1057, 1995)describe a plant overexpressing glutathione reductase (GR), which isresistant to methyl viologen treatment.

Siminszky (Phytochemistry Reviews, 5:445-458, 2006) describes plantcytochrome P450-mediated detoxification of multiple, chemicallyunrelated classes of herbicides.

Other examples of herbicide resistance have been described, forinstance, in U.S. Pat. Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114;6,107,549; 5,866,775; 5,804,425; 5,633,435; 5,463,175.

C. Waxy Starch

The waxy characteristic is an example of a recessive trait. In thisexample, the progeny resulting from the first backcross generation (BC1)must be grown and selfed. A test is then run on the selfed seed from theBC1 plant to determine which BC1 plants carried the recessive gene forthe waxy trait. In other recessive traits additional progeny testing,for example growing additional generations such as the BC1S1, may berequired to determine which plants carry the recessive gene.

D. Disease and Pest Resistance

Plant defenses are often activated by specific interaction between theproduct of a disease resistance gene (R) in the plant and the product ofa corresponding avirulence (Avr) gene in the pathogen. A plant line canbe transformed with cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example, Jones et al.(Science, 266:7891, 1994) (cloning of the tomato Cf-9 gene forresistance to Cladosporium fulvum); Martin et al. (Science, 262: 1432,1993) (tomato Pto gene for resistance to Pseudomonas syringae pv.tomato); and Mindrinos et al. (Cell, 78(6):1089-1099, 1994) (ArabidopsisRPS2 gene for resistance to Pseudomonas syringae).

A viral-invasive protein or a complex toxin derived therefrom may alsobe used for viral disease resistance. For example, the accumulation ofviral coat proteins in transformed plant cells imparts resistance toviral infection and/or disease development effected by the virus fromwhich the coat protein gene is derived, as well as by related viruses.See Beachy et al., Ann. Rev. Phytopathol., 28:451, 1990. Coatprotein-mediated resistance has been conferred upon transformed plantsagainst alfalfa mosaic virus, cucumber mosaic virus, tobacco streakvirus, potato virus X, potato virus Y, tobacco etch virus, tobaccorattle virus and tobacco mosaic virus. Id.

A virus-specific antibody may also be used. See, for example,Tavladoraki et al. (Nature, 366:469, 1993), who show that transgenicplants expressing recombinant antibody genes are protected from virusattack. Virus resistance has also been described in, for example, U.S.Pat. Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023 and5,304,730. Additional means of inducing whole-plant resistance to apathogen include modulation of the systemic acquired resistance (SAR) orpathogenesis related (PR) genes, for example, genes homologous to theArabidopsis thaliana NIM1/NPR1/SAI1, and/or by increasing salicylic acidproduction (Ryals et al., Plant Cell, 8:1809-1819, 1996).

Logemann et al. (Biotechnology, 10:305, 1992), for example, disclosetransgenic plants expressing a barley ribosome-inactivating gene havingan increased resistance to fungal disease. Plant defensins may be usedto provide resistance to fungal pathogens (Thomma et al., Planta,216:193-202, 2002). Other examples of fungal disease resistance areprovided in U.S. Pat. Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407;6,215,048; 5,516,671; 5,773,696; 6,121,436; and 6,316,407.

Nematode resistance has been described, for example, in U.S. Pat. No.6,228,992 and bacterial disease resistance in U.S. Pat. No. 5,516,671.

E. Insect Resistance

One example of an insect resistance gene includes a Bacillusthuringiensis (Bt) protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al. (Gene,48(1):109-118, 1986), who disclose the cloning and nucleotide sequenceof a Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from the American Type Culture Collection,Manassas, Va., for example, under ATCC Accession Nos. 40098, 67136,31995 and 31998. Another example is a lectin. See, for example, VanDamme et al. (Plant Molec. Biol., 24:25, 1994), who disclose thenucleotide sequences of several Clivia miniata mannose-binding lectingenes. A vitamin-binding protein may also be used, such as avidin. SeePCT application US93/06487, the contents of which are herebyincorporated by reference. This application teaches the use of avidinand avidin homologues as larvicides against insect pests.

Yet another insect resistance gene is an enzyme inhibitor, for example,a protease or proteinase inhibitor or an amylase inhibitor. See, forexample, Abe et al., J. Biol. Chem., 262:16793, 1987 (nucleotidesequence of rice cysteine proteinase inhibitor); Huub et al., PlantMolec. Biol., 21:985, 1993 (nucleotide sequence of cDNA encoding tobaccoproteinase inhibitor I); and Sumitani et al., Biosci. Biotech. Biochem.,57:1243, 1993 (nucleotide sequence of Streptomyces nitrosporeusα-amylase inhibitor).

An insect-specific hormone or pheromone may also be used. See, forexample, the disclosure by Hammock et al. (Nature, 344:458, 1990), ofbaculovirus expression of cloned juvenile hormone esterase, aninactivator of juvenile hormone; Gade and Goldsworthy (Eds.Physiological System in Insects, Elsevier Academic Press, Burlington,Mass., 2007), describing allostatins and their potential use in pestcontrol; and Palli et al., Vitam. Horm., 73:59-100, 2005, disclosing useof ecdysteroid and ecdysteroid receptor in agriculture. The diuretichormone receptor (DHR) was identified in Price et al. (Insect Mol.Biol., 13:469-480, 2004) as a candidate target of insecticides.

Still other examples include an insect-specific antibody or animmunotoxin derived therefrom and a developmental-arrestive protein. SeeTaylor et al. (Seventh Inn Symposium on Molecular Plant-MicrobeInteractions, Edinburgh, Scotland, Abstract W97, 1994), who describedenzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments. Numerous other examples of insectresistance have been described. See, for example, U.S. Pat. Nos.6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030;6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756;6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949;6,281,016; 6,248,536; 6,242,241; 6,221,649; 6,177,615; 6,156,573;6,153,814; 6,110,464; 6,093,695; 6,063,756; 6,063,597; 6,023,013;5,959,091; 5,942,664; 5,942,658, 5,880,275; 5,763,245 and 5,763,241.

F. Modified Fatty Acid, Phytate and Carbohydrate Metabolism

Genes may be used conferring modified fatty acid metabolism, in terms ofcontent and quality. For example, stearyl-ACP desaturase genes may beused. See Knutzon et al., Proc. Natl. Acad. Sci. USA, 89:2624, 1992.Various fatty acid desaturases have also been described, such as aSaccharomyces cerevisiae OLE1 gene encoding Δ9-fatty acid desaturase, anenzyme which forms the monounsaturated palmitoleic (16:1) and oleic(18:1) fatty acids from palmitoyl (16:0) or stearoyl (18:0) CoA(McDonough et al., J. Biol. Chem., 267(9):5931-5936, 1992); a geneencoding a stearoyl-acyl carrier protein delta-9 desaturase from castor(Fox et al., Proc. Natl. Acad. Sci. USA, 90(6):2486-2490, 1993); Δ6- andΔ1-desaturases from the cyanobacteria Synechocystis responsible for theconversion of linoleic acid (18:2) to gamma-linolenic acid (18:3 gamma)(Reddy et al., Plant Mol. Biol., 22(2):293-300, 1993); a gene fromArabidopsis thaliana that encodes an omega-3 desaturase (Arondel et al.,Science, 258(5086):1353-1355 1992); plant Δ9-desaturases (PCTApplication Publ. No. WO 91/13972) and soybean and BrassicaΔ15-desaturases (European Patent Application Publ. No. EP 0616644).

Modified oils production is disclosed, for example, in U.S. Pat. Nos.6,444,876; 6,426,447 and 6,380,462. High oil production is disclosed,for example, in U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008 and6,476,295. Modified fatty acid content is disclosed, for example, inU.S. Pat. Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849;6,596,538; 6,589,767; 6,537,750; 6,489,461 and 6,459,018.

Phytate metabolism may also be modified by introduction of aphytase-encoding gene to enhance breakdown of phytate, adding more freephosphate to the transformed plant. For example, see Van Hartingsveldtet al., Gene, 127:87, 1993, for a disclosure of the nucleotide sequenceof an Aspergillus niger phytase gene. In corn, this, for example, couldbe accomplished by cloning and then reintroducing DNA associated withthe single allele which is responsible for corn mutants characterized bylow levels of phytic acid. See Raboy et al., Plant Physiol.,124(1):355-368, 2000.

A number of genes are known that may be used to alter carbohydratemetabolism. For example, plants may be transformed with a gene codingfor an enzyme that alters the branching pattern of starch. See Shirozaet al., J. Bacteriol., 170:810, 1988 (nucleotide sequence ofStreptococcus mutans fructosyltransferase gene); Steinmetz et al., Mol.Gen. Genet., 20:220, 1985 (nucleotide sequence of Bacillus subtilislevansucrase gene); Pen et al., Biotechnology, 10:292, 1992 (productionof transgenic plants that express Bacillus licheniformis α-amylase);U.S. Pat. No. 6,166,292 (low raffinose); Elliot et al., Plant Molec.Biol., 21:515, 1993 (nucleotide sequences of tomato invertase genes);Sergaard et al., J. Biol. Chem., 268:22480, 1993 (site-directedmutagenesis of barley α-amylase gene); Fisher et al., Plant Physiol.,102:1045, 1993 (maize endosperm starch branching enzyme II); and U.S.Pat. Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876 and 6,476,295(starch content). The Z10 gene encoding a 10 kD zein storage proteinfrom maize may also be used to alter the quantities of 10 kD zein in thecells relative to other components (Kirihara et al., Gene,71(2):359-370, 1988).

U.S. Patent Appl. Pub. No. 20030163838 describes maize cellulosesynthase genes and methods of use thereof.

G. Resistance to Abiotic Stress

Abiotic stress includes dehydration or other osmotic stress, salinity,high or low light intensity, high or low temperatures, submergence,exposure to heavy metals, and oxidative stress.Delta-pyrroline-5-carboxylate synthetase (P5CS) from mothbean has beenused to provide protection against general osmotic stress.Mannitol-1-phosphate dehydrogenase (mt1D) from E. coli has been used toprovide protection against drought and salinity. Choline oxidase (codAfrom Arthrobactor globiformis) can protect against cold and salt. E.coli choline dehydrogenase (betA) provides protection against salt.Additional protection from cold can be provided by omega-3-fatty aciddesaturase (fad7) from Arabidopsis thaliana. Trehalose-6-phosphatesynthase and levan sucrase (SacB) from yeast and Bacillus subtilis,respectively, can provide protection against drought (summarized fromAnnex II Genetic Engineering for Abiotic Stress Tolerance in Plants,Consultative Group On International Agricultural Research TechnicalAdvisory Committee). Overexpression of superoxide dismutase can be usedto protect against superoxides, as described in U.S. Pat. No. 5,538,878to Thomas et al.

H. Additional Traits

Additional traits can be introduced into the corn variety of the presentinvention. A non-limiting example of such a trait is a coding sequencewhich decreases RNA and/or protein levels. The decreased RNA and/orprotein levels may be achieved through RNAi methods, such as thosedescribed in U.S. Pat. No. 6,506,559 to Fire and Mellow.

Another trait that may find use with the corn variety of the inventionis a sequence which allows for site-specific recombination. Examples ofsuch sequences include the FRT sequence, used with the FLP recombinase(Zhu and Sadowski, J. Biol. Chem., 270:23044-23054, 1995); and the LOXsequence, used with CRE recombinase (Sauer, Mol. Cell. Biol.,7:2087-2096, 1987). The recombinase genes can be encoded at any locationwithin the genome of the corn plant, and are active in the hemizygousstate.

It may also be desirable to make corn plants more tolerant to or moreeasily transformed with Agrobacterium tumefaciens. Expression of p53 andiap, two baculovirus cell-death suppressor genes, inhibited tissuenecrosis and DNA cleavage. Additional targets can include plant-encodedproteins that interact with the Agrobacterium Vir genes; enzymesinvolved in plant cell wall formation; and histones, histoneacetyltransferases and histone deacetylases (reviewed in Gelvin,Microbiology & Mol. Biol. Reviews, 67:16-37, 2003).

In addition to the modification of oil, fatty acid or phytate contentdescribed above, it may additionally be beneficial to modify the amountsor levels of other compounds. For example, the amount or composition ofantioxidants can be altered. See, for example, U.S. Pat. No. 6,787,618,U.S. Patent Appl. Pub. No. 20040034886 and International Patent Appl.Pub. No. WO 00/68393, which disclose the manipulation of antioxidantlevels, and International Patent Appl. Pub. No. WO 03/082899, whichdiscloses the manipulation of a antioxidant biosynthetic pathway.

Additionally, seed amino acid content may be manipulated. U.S. Pat. No.5,850,016 and International Patent Appl. Pub. No. WO 99/40209 disclosethe alteration of the amino acid compositions of seeds. U.S. Pat. Nos.6,080,913 and 6,127,600 disclose methods of increasing accumulation ofessential amino acids in seeds.

U.S. Pat. No. 5,559,223 describes synthetic storage proteins in whichthe levels of essential amino acids can be manipulated. InternationalPatent Appl. Pub. No. WO 99/29882 discloses methods for altering aminoacid content of proteins. International Patent Appl. Pub. No. WO98/20133 describes proteins with enhanced levels of essential aminoacids. International Patent Appl. Pub. No. WO 98/56935 and U.S. Pat.Nos. 6,346,403, 6,441,274 and 6,664,445 disclose plant amino acidbiosynthetic enzymes. International Patent Appl. Pub. No. WO 98/45458describes synthetic seed proteins having a higher percentage ofessential amino acids than wildtype.

U.S. Pat. No. 5,633,436 discloses plants comprising a higher content ofsulfur-containing amino acids; U.S. Pat. No. 5,885,801 discloses plantscomprising a high threonine content; U.S. Pat. Nos. 5,885,802 and5,912,414 disclose plants comprising high methionine content; U.S. Pat.No. 5,990,389 discloses plants comprising a high lysine content; U.S.Pat. No. 6,459,019 discloses plants comprising an increased lysine andthreonine content; International Patent Appl. Pub. No. WO 98/42831discloses plants comprising a high lysine content; International PatentAppl. Pub. No. WO 96/01905 discloses plants comprising a high threoninecontent; and International Patent Appl. Pub. No. WO 95/15392 disclosesplants comprising a high lysine content.

I. Origin and Breeding History of an Exemplary Introduced Trait

85DGD1 MLms is a conversion of 85DGD1 to cytoplasmic male sterility.85DGD1 MLms was derived using backcross methods. 85DGD1 (a proprietaryinbred of Monsanto Company) was used as the recurrent parent and MLms, agermplasm source carrying ML cytoplasmic sterility, was used as thenonrecurrent parent. The breeding history of the converted inbred 85DGD1MLms can be summarized as follows:

Hawaii Nurseries Made up S-O: Female row 585 male Planting Date Apr. 2,1992 row 500 Hawaii Nurseries S-O was grown and plants were PlantingDate Jul. 15, 1992 backcrossed times 85DGD1 (rows 444 {acute over ( )}443) Hawaii Nurseries Bulked seed of the BC1 was grown and Planting DateNov. 18, 1992 backcrossed times 85DGD1 (rows V3-27 {acute over ( )}V3-26) Hawaii Nurseries Bulked seed of the BC2 was grown and PlantingDate Apr. 2, 1993 backcrossed times 85DGD1 (rows 37 {acute over ( )} 36)Hawaii Nurseries Bulked seed of the BC3 was grown and Planting Date Jul.14, 1993 backcrossed times 85DGD1 (rows 99 {acute over ( )} 98) HawaiiNurseries Bulked seed of BC4 was grown and Planting Date Oct. 28, 1993backcrossed times 85DGD1 (rows KS-63 {acute over ( )} KS-62) Summer 1994A single ear of the BC5 was grown and backcrossed times 85DGD1 (MC94-822{acute over ( )} MC94-822-7) Winter 1994 Bulked seed of the BC6 wasgrown and backcrossed times 85DGD1 (3Q-1 {acute over ( )} 3Q-2) Summer1995 Seed of the BC7 was bulked and named 85DGD1 MLms.

J. Illustrative Procedures for Introduction of a Desired Trait

As described above, techniques for the production of corn plants withadded traits are well known in the art (see, e.g., Poehlman et al., In:Breeding Field Crops, 4^(th) Ed., Iowa State University Press, Ames,Iowa, 132-155; 321-344, 1995; Fehr, Principles of Cultivar Development,1:360-376, 1987; Sprague and Dudley, In: Corn and Corn Improvement,3^(rd) Ed., Crop Science of America, Inc.; Soil Science of America,Inc., Wisconsin. 881-883; 901-918, 1988). A non-limiting example of sucha procedure one of skill in the art would use for preparation of a cornplant of CV032539 comprising an added trait is as follows:

-   -   (a) crossing corn plant CV032539 to a second (nonrecurrent) corn        plant comprising a locus to be converted in corn plant CV032539;    -   (b) selecting at least a first progeny plant resulting from the        crossing and comprising the locus;    -   (c) crossing the selected progeny to corn plant CV032539; and    -   (d) repeating steps (b) and (c) until a plant of variety        CV032539 is obtained comprising the locus.

Following these steps, essentially any locus may be introduced into cornvariety CV032539. For example, molecular techniques allow introductionof any given locus, without the need for phenotypic screening of progenyduring the backcros sing steps.

PCR™ and Southern hybridization are two examples of molecular techniquesthat may be used for confirmation of the presence of a given locus andthus conversion of that locus. The techniques are carried out asfollows: Seeds of progeny plants are grown and DNA isolated from leaftissue (see Sambrook et al., In: Molecular cloning, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2001; Shure et al., Cell,35(1):225-233, 1983). Approximately one gram of leaf tissue islyophilized overnight in 15 ml polypropylene tubes. Freeze-dried tissueis ground to a powder in the tube using a glass rod. Powdered tissue ismixed thoroughly with 3 ml extraction buffer (7.0M urea, 0.35M NaCl,0.05M Tris-HCl pH 8.0, 0.01M EDTA, 1% sarcosine). Tissue/bufferhomogenate is extracted with 3 ml phenol/chloroform. The aqueous phaseis separated by centrifugation, and precipitated twice using 1/10 volumeof 4.4M ammonium acetate, pH 5.2, and an equal volume of isopropanol.The precipitate is washed with 75% ethanol and resuspended in 100-500 μlTE (0.01M Tris-HCl, 0.001M EDTA, pH 8.0). The DNA may then be screenedas desired for presence of the locus.

For PCR™, 200-1000 ng genomic DNA from the progeny plant being screenedis added to a reaction mix containing 10 mM Tris-HCl, pH 8.3, 1.5 mMMgCl₂, 50 mM KCl, 0.1 mg/ml gelatin, 200 μM each dATP, dCTP, dGTP, dTTP,20% glycerol, 2.5 units Taq DNA polymerase and 0.5 μM each of forwardand reverse DNA primers that span a segment of the locus beingconverted. The reaction is run in a thermal cycling machine 3 minutes at94 C, 39 repeats of the cycle 1 minute at 94 C, 1 minute at 50 C, 30seconds at 72° C., followed by 5 minutes at 72° C. Twenty μl of eachreaction mix is run on a 3.5% NuSieve gel in TBE buffer (90 mMTris-borate, 2 mM EDTA) at 50V for two to four hours. The amplifiedfragment is detected using an agarose gel. Detection of an amplifiedfragment corresponding to the segment of the locus spanned by theprimers indicates the presence of the locus.

For Southern analysis, plant DNA is restricted, separated in an agarosegel and transferred to a Nylon filter in 10×SCP (20 SCP: 2M NaCl, 0.6Mdisodium phosphate, 0.02M disodium EDTA) according to standard methods(Southern, J. Mol. Biol., 98:503-517, 1975). Locus DNA or RNA sequencesare labeled, for example, radioactively with ³²P by random priming(Feinberg & Vogelstein, Anal. Biochem., 132(1):6-13, 1983). Filters areprehybridized in 6×SCP, 10% dextran sulfate, 2% sarcosine, and 500 μg/mldenatured salmon sperm DNA. The labeled probe is denatured, hybridizedto the filter and washed in 2×SCP, 1% SDS at 65° C. for 30 minutes andvisualized by autoradiography using Kodak XAR5 film. Presence of thelocus is indicated by detection of restriction fragments of theappropriate size.

V. Tissue Cultures and In Vitro Regeneration of Corn Plants

A further aspect of the invention relates to tissue cultures of the cornplant designated CV032539. As used herein, the term “tissue culture”indicates a composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant. Exemplary types of tissue cultures are protoplasts, calli andplant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, kernels, ears, cobs, leaves, husks, stalks,roots, root tips, anthers, silk, and the like. In a preferredembodiment, the tissue culture comprises embryos, protoplasts,meristematic cells, pollen, leaves or anthers derived from immaturetissues of these plant parts. Means for preparing and maintaining planttissue cultures are well known in the art (U.S. Pat. No. 5,538,880; andU.S. Pat. No. 5,550,318, each incorporated herein by reference in theirentirety). By way of example, a tissue culture comprising organs such astassels or anthers has been used to produce regenerated plants (U.S.Pat. No. 5,445,961 and U.S. Pat. No. 5,322,789; the disclosures of whichare incorporated herein by reference).

One type of tissue culture is tassel/anther culture. Tassels containanthers which in turn enclose microspores. Microspores develop intopollen. For anther/microspore culture, if tassels are the plantcomposition, they are preferably selected at a stage when themicrospores are uninucleate, that is, include only one, rather than 2 or3 nuclei. Methods to determine the correct stage are well known to thoseskilled in the art and include mitramycin fluorescent staining (Pace etal., Theoretical and Applied Genetics, 73:863-869, 1987), trypan blue(preferred) and acetocarmine squashing. The mid-uninucleate microsporestage has been found to be the developmental stage most responsive tothe subsequent methods disclosed to ultimately produce plants.

Although microspore-containing plant organs such as tassels cangenerally be pretreated at any cold temperature below about 25° C., arange of 4 to 25° C. is preferred, and a range of 8 to 14° C. isparticularly preferred. Although other temperatures yield embryoids andregenerated plants, cold temperatures produce optimum response ratescompared to pretreatment at temperatures outside the preferred range.Response rate is measured as either the number of embryoids or thenumber of regenerated plants per number of microspores initiated inculture. Exemplary methods of microspore culture are disclosed in, forexample, Gaillard et al., Plant Cell Reports, 10(2):55, 1991, and U.S.Pat. No. 5,322,789 and U.S. Pat. No. 5,445,961, the disclosures of whichare specifically incorporated herein by reference.

Although not required, when tassels are employed as the plant organ, itis generally preferred to sterilize their surface. Following surfacesterilization of the tassels, for example, with a solution of calciumhypochloride, the anthers are removed from about 70 to 150 spikelets(small portions of the tassels) and placed in a preculture orpretreatment medium. Larger or smaller amounts can be used depending onthe number of anthers.

When one elects to employ tassels directly, tassels are preferablypretreated at a cold temperature for a predefined time, preferably at10° C. for about 4 days. After pretreatment of a whole tassel at a coldtemperature, dissected anthers are further pretreated in an environmentthat diverts microspores from their developmental pathway. The functionof the preculture medium is to switch the developmental program from oneof pollen development to that of embryoid/callus development. Anembodiment of such an environment in the form of a preculture mediumincludes a sugar alcohol, for example mannitol or sorbitol, inositol orthe like. An exemplary synergistic combination is the use of mannitol ata temperature of about 10° C. for a period ranging from about 10 to 14days. In a preferred embodiment, 3 ml of 0.3M mannitol combined with 50mg/l of ascorbic acid, silver nitrate, and colchicine is used forincubation of anthers at 10° C. for between 10 and 14 days. Anotherembodiment is to substitute sorbitol for mannitol. The colchicineproduces chromosome doubling at this early stage. The chromosomedoubling agent is preferably only present at the preculture stage.

It is believed that the mannitol or other similar carbon structure orenvironmental stress induces starvation and functions to forcemicrospores to focus their energies on entering developmental stages.The cells are unable to use, for example, mannitol as a carbon source atthis stage. It is believed that these treatments confuse the cellscausing them to develop as embryoids and plants from microspores.Dramatic increases in development from these haploid cells, as high as25 embryoids in 10⁴ microspores, have resulted from using these methods.

To isolate microspores, an isolation media is preferred. An isolationmedia is used to separate microspores from the anther walls whilemaintaining their viability and embryogenic potential. An illustrativeembodiment of an isolation media includes a 6% sucrose or maltosesolution combined with an antioxidant such as 50 mg/l of ascorbic acid,0.1 mg/l biotin, and 400 mg/l of proline, combined with 10 mg/l ofnicotinic acid and 0.5 mg/l AgNO₃. In another embodiment, the biotin andproline are omitted.

An isolation media preferably has a higher antioxidant level where it isused to isolate microspores from a donor plant (a plant from which aplant composition containing a microspore is obtained) that is fieldgrown in contrast to greenhouse grown. A preferred level of ascorbicacid in an isolation medium is from about 50 mg/l to about 125 mg/l and,more preferably, from about 50 mg/l to about 100 mg/l.

One can find particular benefit in employing a support for themicrospores during culturing and subculturing. Any support thatmaintains the cells near the surface can be used. An illustrativeembodiment of a solid support is a TRANSWELL® culture dish. Anotherembodiment of a solid support for development of the microspores is abilayer plate wherein liquid media is on top of a solid base. Otherembodiments include a mesh or a millipore filter. Preferably, a solidsupport is a nylon mesh in the shape of a raft. A raft is defined as anapproximately circular support material which is capable of floatingslightly above the bottom of a tissue culture vessel, for example, apetri dish, of about a 60 or 100 mm size, although any other laboratorytissue culture vessel will suffice. In an illustrative embodiment, araft is about 55 mm in diameter.

Culturing isolated microspores on a solid support, for example, on a 10mm pore nylon raft floating on 2.2 ml of medium in a 60 mm petri dish,prevents microspores from sinking into the liquid medium and thusavoiding low oxygen tension. These types of cell supports enable theserial transfer of the nylon raft with its associatedmicrospore/embryoids ultimately to full strength medium containingactivated charcoal and solidified with, for example, GELRITE™(solidifying agent).

The liquid medium passes through the mesh while the microspores areretained and supported at the medium-air interface. The surface tensionof the liquid medium in the petri dish causes the raft to float. Theliquid is able to pass through the mesh; consequently, the microsporesstay on top. The mesh remains on top of the total volume of liquidmedium.

The culture vessels can be further defined as either (1) a bilayer 60 mmpetri plate wherein the bottom 2 ml of medium are solidified with 0.7%agarose overlaid with 1 mm of liquid containing the microspores; (2) anylon mesh raft wherein a wafer of nylon is floated on 1.2 ml of mediumand 1 ml of isolated microspores is pipetted on top; or (3) TRANSWELL®plates wherein isolated microspores are pipetted onto membrane insertswhich support the microspores at the surface of 2 ml of medium.

Examples of processes of tissue culturing and regeneration of corn aredescribed in, for example, European Patent Application 0 160 390, Greenand Rhodes, Maize for Biological Research, 367-372, 1982; and Duncan etal., Planta, 165:322-332, 1985; Songstad et al., Plant Cell Reports,7:262-265, 1988; Rao et al., In: Somatic Embryogenesis in Glume CallusCultures, Maize Genetics Cooperation Newsletter #60, 1986; Conger etal., Plant Cell Reports, 6:345-347, 1987; PCT Application WO 95/06128,Armstrong and Green, Planta, 164:207-214, 1985; Gordon-Kamm et al., ThePlant Cell, 2:603-618, 1990; Gaillard et al., Plant Cell Reports,10(2):55, 1991; and U.S. Pat. No. 5,736,369.

VI. Processes of Crossing Corn Plants and the Corn Plants Produced bySuch Crosses

The present invention provides processes of preparing novel corn plantsand corn plants produced by such processes. In accordance with such aprocess, a first parent corn plant may be crossed with a second parentcorn plant wherein at least one of the first and second corn plants isthe inbred corn plant CV032539. One application of the process is in theproduction of F₁ hybrid plants. Another important aspect of this processis that it can be used for the development of novel inbred lines. Forexample, the inbred corn plant CV032539 could be crossed to any secondplant, and the resulting hybrid progeny each selfed for about 5 to 7 ormore generations, thereby providing a large number of distinct,pure-breeding inbred lines. These inbred lines could then be crossedwith other inbred or non-inbred lines and the resulting hybrid progenyanalyzed for beneficial characteristics. In this way, novel inbred linesconferring desirable characteristics could be identified.

Corn plants (Zea mays L.) can be crossed by either natural or mechanicaltechniques. Natural pollination occurs in corn when wind blows pollenfrom the tassels to the silks that protrude from the tops of therecipient ears. Mechanical pollination can be effected either bycontrolling the types of pollen that can blow onto the silks or bypollinating by hand. In one embodiment, crossing comprises the steps of:

-   -   (a) planting in pollinating proximity seeds of a first and a        second parent corn plant, and preferably, seeds of a first        inbred corn plant and a second, distinct inbred corn plant;    -   (b) cultivating or growing the seeds of the first and second        parent corn plants into plants that bear flowers;    -   (c) emasculating flowers of either the first or second parent        corn plant, i.e., treating the flowers so as to prevent pollen        production, or alternatively, using as the female parent a male        sterile plant, thereby providing an emasculated parent corn        plant;    -   (d) allowing natural cross-pollination to occur between the        first and second parent corn plants;    -   (e) harvesting seeds produced on the emasculated parent corn        plant; and, where desired,    -   (f) growing the harvested seed into a corn plant, preferably, a        hybrid corn plant.

Parental plants are typically planted in pollinating proximity to eachother by planting the parental plants in alternating rows, in blocks orin any other convenient planting pattern. Where the parental plantsdiffer in timing of sexual maturity, it may be desired to plant theslower maturing plant first, thereby ensuring the availability of pollenfrom the male parent during the time at which silks on the female parentare receptive to pollen. Plants of both parental parents are cultivatedand allowed to grow until the time of flowering. Advantageously, duringthis growth stage, plants are in general treated with fertilizer and/orother agricultural chemicals as considered appropriate by the grower.

At the time of flowering, in the event that plant CV032539 is employedas the male parent, the tassels of the other parental plant are removedfrom all plants employed as the female parental plant to avoidself-pollination. The detasseling can be achieved manually but also canbe done by machine, if desired. Alternatively, when the female parentcorn plant comprises a cytoplasmic or nuclear gene conferring malesterility, detasseling may not be required. Additionally, a chemicalgametocide may be used to sterilize the male flowers of the femaleplant. In this case, the parent plants used as the male may either notbe treated with the chemical agent or may comprise a genetic factorwhich causes resistance to the emasculating effects of the chemicalagent. Gametocides affect processes or cells involved in thedevelopment, maturation or release of pollen. Plants treated with suchgametocides are rendered male sterile, but typically remain femalefertile. The use of chemical gametocides is described, for example, inU.S. Pat. No. 4,936,904, the disclosure of which is specificallyincorporated herein by reference in its entirety. Furthermore, the useof glyphosate herbicide to produce male sterile corn plants is disclosedin U.S. Pat. No. 6,762,344 and PCT Publication WO 98/44140.

Following emasculation, the plants are then typically allowed tocontinue to grow and natural cross-pollination occurs as a result of theaction of wind, which is normal in the pollination of grasses, includingcorn. As a result of the emasculation of the female parent plant, allthe pollen from the male parent plant is available for pollinationbecause tassels, and thereby pollen bearing flowering parts, have beenpreviously removed from all plants of the inbred plant being used as thefemale in the hybridization. Of course, during this hybridizationprocedure, the parental varieties are grown such that they are isolatedfrom other corn fields to minimize or prevent any accidentalcontamination of pollen from foreign sources. These isolation techniquesare well within the skill of those skilled in this art.

Both parental inbred plants of corn may be allowed to continue to growuntil maturity or the male rows may be destroyed after flowering iscomplete. Only the ears from the female inbred parental plants areharvested to obtain seeds of a novel F₁ hybrid. The novel F₁ hybrid seedproduced can then be planted in a subsequent growing season incommercial fields or, alternatively, advanced in breeding protocols forpurposes of developing novel inbred lines.

Alternatively, in another embodiment of the invention, both first andsecond parent corn plants can be from variety CV032539. Thus, any cornplant produced using corn plant CV032539 forms a part of the invention.As used herein, crossing can mean selfing, backcrossing, crossing toanother or the same inbred, crossing to populations, and the like. Allcorn plants produced using the inbred corn plant CV032539 as a parentare, therefore, within the scope of this invention.

A. F₁ Hybrid Corn Plant and Seed Production

One beneficial use of the instant corn variety is in the production ofhybrid seed. Any time the inbred corn plant CV032539 is crossed withanother, different, corn inbred, a first generation (F₁) corn hybridplant is produced. As such, an F₁ hybrid corn plant can be produced bycrossing CV032539 with any second inbred maize plant. Essentially anyother corn plant can be used to produce a hybrid corn plant having cornplant CV032539 as one parent. All that is required is that the secondplant be fertile, which corn plants naturally are, and that the plant isnot corn variety CV032539.

The goal of the process of producing an F₁ hybrid is to manipulate thegenetic complement of corn to generate new combinations of genes whichinteract to yield new or improved traits (phenotypic characteristics). Aprocess of producing an F₁ hybrid typically begins with the productionof one or more inbred plants. Those plants are produced by repeatedcrossing of ancestrally related corn plants to try to combine certaingenes within the inbred plants.

Corn has a diploid phase which means two conditions of a gene (twoalleles) occupy each locus (position on a chromosome). If the allelesare the same at a locus, there is said to be homozygosity. If they aredifferent, there is said to be heterozygosity. In a completely inbredplant, all loci are homozygous. Because many loci when homozygous aredeleterious to the plant, in particular leading to reduced vigor, lesskernels, weak and/or poor growth, production of inbred plants is anunpredictable and arduous process. Under some conditions, heterozygousadvantage at some loci effectively bars perpetuation of homozygosity.

A single cross hybrid corn variety is the cross of two inbred plants,each of which has a genotype which complements the genotype of theother. Typically, F₁ hybrids are more vigorous than their inbredparents. This hybrid vigor, or heterosis, is manifested in manypolygenic traits, including markedly improved yields, better stalks,better roots, better uniformity and better insect and diseaseresistance. In the development of hybrids only the F₁ hybrid plants aretypically sought. An F₁ single cross hybrid is produced when two inbredplants are crossed. A double cross hybrid is produced from four inbredplants crossed in pairs (A×B and C×D) and then the two F₁ hybrids arecrossed again (A×B)×(C×D).

Thousands of corn varieties are known to those of skill in the art, anyone of which could be crossed with corn plant CV032539 to produce ahybrid plant. For example, the U.S. Patent & Trademark Office has issuedmore than 300 utility patents for corn varieties. Estimates place thenumber of different corn accessions in gene banks around the world ataround 50,000 (Chang, In Plant Breeding in the 1990s, Stalker and Murphy(Eds.), Wallingford, U.K., CAB International, 17-35, 1992). The MaizeGenetics Cooperation Stock Center, which is supported by the U.S.Department of Agriculture, has a total collection approaching 80,000individually pedigreed samples (available on the world wide web atmaizecoop.cropsci.uiuc.edu/).

An example of an F₁ hybrid which has been produced with CV032539 as aparent is the hybrid CH070904. Hybrid CH070904 was produced by crossinginbred corn plant CV032539 with the inbred corn plant designatedCV342520 (U.S. Pat. No. 8,981,189, the disclosure of which isspecifically incorporated herein by reference in its entirety).

When the inbred corn plant CV032539 is crossed with another inbred plantto yield a hybrid, it can serve as either the maternal or paternalplant. For many crosses, the outcome is the same regardless of theassigned sex of the parental plants. Depending on the seed productioncharacteristics relative to a second parent in a hybrid cross, it may bedesired to use one of the parental plants as the male or female parent.Some plants produce tighter ear husks leading to more loss, for exampledue to rot. There can be delays in silk formation which deleteriouslyaffect timing of the reproductive cycle for a pair of parental plants.Seed coat characteristics can be preferable in one plant. Pollen can beshed better by one plant. Therefore, a decision to use one parent plantas a male or female may be made based on any such characteristics as iswell known to those of skill in the art.

B. Development of Corn Varieties

The development of new varieties using one or more starting varieties iswell known in the art. In accordance with the invention, novel varietiesmay be created by crossing corn variety CV032539 followed by multiplegenerations of breeding according to such well known methods. Newvarieties may be created by crossing corn variety CV032539 with anysecond plant. In selecting such a second plant to cross for the purposeof developing novel inbred lines, it may be desired to choose thoseplants which either themselves exhibit one or more selected desirablecharacteristics or which exhibit the desired characteristic(s) when inhybrid combination. Examples of potentially desired characteristicsinclude greater yield, better stalks, better roots, resistance toinsecticides, herbicides, pests, and disease, tolerance to heat anddrought, reduced time to crop maturity, better agronomic quality, highernutritional value, and uniformity in germination times, standestablishment, growth rate, maturity, and fruit size.

Once initial crosses have been made with corn variety CV032539,inbreeding takes place to produce new inbred varieties. Inbreedingrequires manipulation by human breeders. Even in the extremely unlikelyevent inbreeding rather than crossbreeding occurred in natural corn,achievement of complete inbreeding cannot be expected in nature due towell known deleterious effects of homozygosity and the large number ofgenerations the plant would have to breed in isolation. The reason forthe breeder to create inbred plants is to have a known reservoir ofgenes whose gametic transmission is predictable.

The pedigree breeding method involves crossing two genotypes. Eachgenotype can have one or more desirable characteristics lacking in theother; or, each genotype can complement the other. If the two originalparental genotypes do not provide all of the desired characteristics,other genotypes can be included in the breeding population. Superiorplants that are the products of these crosses are selfed and selected insuccessive generations. Each succeeding generation becomes morehomogeneous as a result of self-pollination and selection. Typically,this method of breeding involves five or more generations of selfing andselection: S₁→S₂; S₂→S₃; S₃→S₄; S₄→S₅, etc. After at least fivegenerations, the inbred plant is considered genetically pure.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny are heterozygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other loci. The last backcrossgeneration would be selfed to give pure breeding progeny for the traitbeing transferred.

The development of a hybrid corn variety involves three steps: (1) theselection of plants from various germplasm pools; (2) the selfing of theselected plants for several generations to produce a series of inbredplants, which, although different from each other, each breed true andare highly uniform; and (3) crossing the selected inbred plants withunrelated inbred plants to produce the hybrid progeny (F₁). During theinbreeding process in corn, the vigor of the plants decreases. Vigor isrestored when two unrelated inbred plants are crossed to produce thehybrid progeny (F₁). An important consequence of the homozygosity andhomogeneity of the inbred plants is that the hybrid between any twoinbreds is always the same. Once the inbreds that give a superior hybridhave been identified, hybrid seed can be reproduced indefinitely as longas the homogeneity of the inbred parents is maintained. Conversely, muchof the hybrid vigor exhibited by F₁ hybrids is lost in the nextgeneration (F₂). Consequently, seed from hybrid varieties is not usedfor planting stock. It is not generally beneficial for farmers to saveseed of F₁ hybrids. Rather, farmers purchase F₁ hybrid seed for plantingevery year.

The development of inbred plants generally requires at least about 5 to7 generations of selfing. Inbred plants are then cross-bred in anattempt to develop improved F₁ hybrids. Hybrids are then screened andevaluated in small scale field trials. Typically, about 10 to 15phenotypic traits, selected for their potential commercial value, aremeasured. A selection index of the most commercially important traits isused to help evaluate hybrids. FACT, an acronym for Field AnalysisComparison Trial (strip trials), is an on-farm experimental testingprogram employed by Monsanto Company to perform the final evaluation ofthe commercial potential of a product.

During the next several years, a progressive elimination of hybridsoccurs based on more detailed evaluation of their phenotype. Eventually,strip trials (FACT) are conducted to formally compare the experimentalhybrids being developed with other hybrids, some of which werepreviously developed and generally are commercially successful. That is,comparisons of experimental hybrids are made to competitive hybrids todetermine if there was any advantage to further development of theexperimental hybrids. Examples of such comparisons are presented hereinbelow. After FACT testing is complete, determinations may be madewhether commercial development should proceed for a given hybrid.

C. F₁ Hybrid Comparisons

As mentioned above, hybrids are progressively eliminated followingdetailed evaluations of their phenotype, including formal comparisonswith other commercially successful hybrids. Strip trials are used tocompare the phenotypes of hybrids grown in as many environments aspossible. They are performed in many environments to assess overallperformance of the new hybrids and to select optimum growing conditions.Because the corn is grown in close proximity, environmental factors thataffect gene expression, such as moisture, temperature, sunlight, andpests, are minimized. For a decision to be made to commercialize ahybrid, it is not necessary that the hybrid be better than all otherhybrids. Rather, significant improvements must be shown in at least sometraits that would create improvements in some niches.

Examples of such comparative data are set forth herein below in Table 2,which presents a comparison of performance data for a hybrid made withCV032539 as one parent, versus selected hybrids of commercial value.

TABLE 2 Comparative Data for CH070904, a Hybrid Having CV032539 as OneInbred Parent Entries Compared YLD_BE MST STLP RTLP FNSP SDV PHT EHT TWTSTGR GSPP CH070904 159.7 17.69 0.9 0.9 101 4 87.5 37.5 58.7 5.1 0DKC36-26 161.9 16.86 0.4 1 97 4 88.1 39.1 56.2 6.4 0 Deviation −2.210.83 0.45 −0.04 3.92 0 −0.62 −1.62 2.43 −1.33 0 Significance * * ** +Significance levels are indicated as: + = 10%, * = 5%, ** = 1% LEGENDABBREVIATIONS: YLD_B = Yield (bushels/acre) MST = Moisture STLP = StalkLodging (percent) RTLP = Root Lodging (percent) FNSP = Final Stand(percent of test mean) SDV = Seedling Vigor Rating PHT = Plant Height(inches) EHT = Ear Height (inches) TWT = Test Weight (pounds) STG =Staygreen Rating GSPP = Greensnap (percentage) GDU = GDUs to Shed SLK =GDUs to Silk

D. Physical Description of F₁ Hybrids

The present invention provides F₁ hybrid corn plants derived from thecorn plant CV032539. The physical characteristics of an exemplary hybridproduced using CV032539 as one inbred parent are set forth in Table 3.An explanation of terms used in Table 3 can be found in the Definitions,set forth hereinabove.

TABLE 3 Morphological Traits for CH070904, a Hybrid Having CV032539 asOne Inbred Parent CHARACTERISTIC VALUE 1. STALK Plant Height cm. 261.0Ear Height cm. 72.8 Anthocyanin Absent Brace Root Color Faint InternodeDirection Straight Internode Length cm. 19.7 2. LEAF Color Dark GreenLength cm. 83.3 Width cm. 9.3 Sheath Anthocyanin Absent SheathPubescence Light Marginal Waves Few Longitudinal Creases Many 3. TASSELLength cm. 43.7 Peduncle Length cm. 10.5 Branch Number 8.1 Anther ColorYellow Glume Color Purple Glume Band Absent 4. EAR Silk Color YellowNumber Per Stalk 1.0 Position (attitude) Horizontal Length cm. 17.5Shape Semi-Conical Diameter cm. 4.7 Shank Length cm. 14.2 Husk BractShort Husk Cover cm. 4.8 Husk Opening Tight Husk Color Fresh Green HuskColor Dry Buff Cob Diameter cm. 2.4 Cob Color Red Shelling Percent 88.05. KERNEL Number Per Row 33.6 Row Direction Straight Type Dent Cap ColorDeep Yellow Side Color Yellow Length (depth) mm. 12.8 Width mm. 8.3Thickness 4.5 Endosperm Type Normal Endosperm Color Yellow *These aretypical values. Values may vary due to environment. Other values thatare substantially equivalent are also within the scope of the invention.

VII. Genetic Complements

The present invention provides a genetic complement of the inbred cornplant variety designated CV032539. Further provided by the invention isa hybrid genetic complement, wherein the complement is formed by thecombination of a haploid genetic complement from CV032539 and anotherhaploid genetic complement. Means for determining such a geneticcomplement are well-known in the art.

As used herein, the phrase “genetic complement” means an aggregate ofnucleotide sequences, the expression of which defines the phenotype of acorn plant or a cell or tissue of that plant. By way of example, a cornplant is genotyped to determine a representative sample of the inheritedmarkers it possesses. Markers are alleles at a single locus. They arepreferably inherited in codominant fashion so that the presence of bothalleles at a diploid locus is readily detectable, and they are free ofenvironmental variation, i.e., their heritability is 1. This genotypingis preferably performed on at least one generation of the descendantplant for which the numerical value of the quantitative trait or traitsof interest are also determined. The array of single locus genotypes isexpressed as a profile of marker alleles, two at each locus. The markerallelic composition of each locus can be either homozygous orheterozygous. Homozygosity is a condition where both alleles at a locusare characterized by the same nucleotide sequence or size of a repeatedsequence. Heterozygosity refers to different conditions of the gene at alocus. A preferred type of genetic marker for use with the invention issimple sequence repeats (SSRs), although potentially any other type ofgenetic marker could be used, for example, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),single nucleotide polymorphisms (SNPs), and isozymes.

A genetic marker profile of an inbred may be predictive of the agronomictraits of a hybrid produced using that inbred. For example, if an inbredof known genetic marker profile and phenotype is crossed with a secondinbred of known genetic marker profile and phenotype it is possible topredict the phenotype of the F₁ hybrid based on the combined geneticmarker profiles of the parent inbreds. Methods for prediction of hybridperformance from genetic marker data is disclosed in U.S. Pat. No.5,492,547, the disclosure of which is specifically incorporated hereinby reference in its entirety. Such predictions may be made using anysuitable genetic marker, for example, SSRs, RFLPs, AFLPs, SNPs, orisozymes.

SSRs are genetic markers based on polymorphisms in repeated nucleotidesequences, such as microsatellites. A marker system based on SSRs can behighly informative in linkage analysis relative to other marker systemsin that multiple alleles may be present. Another advantage of this typeof marker is that, through use of flanking primers, detection of SSRscan be achieved, for example, by the polymerase chain reaction (PCR™),thereby eliminating the need for labor-intensive Southern hybridization.The PCR™ detection is done by use of two oligonucleotide primersflanking the polymorphic segment of repetitive DNA. Repeated cycles ofheat denaturation of the DNA followed by annealing of the primers totheir complementary sequences at low temperatures, and extension of theannealed primers with DNA polymerase, comprise the major part of themethodology. Following amplification, markers can be scored by gelelectrophoresis of the amplification products. Scoring of markergenotype is based on the size (number of base pairs) of the amplifiedsegment.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

What is claimed is:
 1. A plant of corn variety CV032539, wherein asample of seed of corn variety CV032539 has been deposited under ATCCAccession No. ______.
 2. A plant part of the plant of claim
 1. 3. Theplant part of claim 2, further defined as pollen, an ovule, or a cell.4. A seed of corn variety CV032539, wherein a sample of seed of cornvariety CV032539 has been deposited under ATCC Accession No. ______. 5.The seed of claim 4, further comprising a transgene, wherein thetransgene was introduced into corn variety CV032539 by backcrossing orgenetic transformation.
 6. A composition comprising the seed of claim 4comprised in plant seed growth media, wherein a sample of seed of cornvariety CV032539 has been deposited under ATCC Accession No. ______. 7.The composition of claim 6, wherein the growth media is soil or asynthetic cultivation medium.
 8. An F₁ hybrid seed produced by crossinga plant of corn variety CV032539 according to claim 1 with a second,distinct corn plant.
 9. The F₁ hybrid seed of claim 8, wherein saidplant of corn variety CV032539 comprises a transgene that is inheritedby the seed, wherein the transgene was introduced into corn varietyCV032539 by backcrossing or genetic transformation.
 10. An F₁ hybridplant grown from the seed of claim
 8. 11. A plant of corn varietyCV032539 further comprising a single locus conversion, wherein a sampleof seed of corn variety CV032539 has been deposited under ATCC AccessionNo. ______.
 12. The plant of claim 11, wherein the single locusconversion comprises a transgene.
 13. A seed that produces the plant ofclaim
 11. 14. The seed of claim 13, wherein the single locus confers atrait selected from the group consisting of male sterility, herbicidetolerance, insect or pest resistance, disease resistance, modified fattyacid metabolism, abiotic stress resistance, altered seed amino acidcomposition, site-specific genetic recombination, and modifiedcarbohydrate metabolism.
 15. The seed of claim 13, wherein the herbicidetolerance trait is tolerance to an herbicide selected from the groupconsisting of glyphosate, sulfonylurea, imidazalinone, dicamba,glufosinate, phenoxy propionic acid, cyclohexanedione, triazine,benzonitrile, PPO-inhibitor herbicides and bromoxynil.
 16. A method ofproducing a seed of a progeny plant derived from the corn varietyCV032539, the method comprising crossing a plant of corn varietyCV032539 according to claim 1 with a corn plant of a second variety toproduce at least a first seed.
 17. The method of claim 16, furthercomprising the steps of (a) crossing a plant grown from said seed withitself or a second plant to produce a seed of a progeny plant of asubsequent generation; (b) growing a progeny plant of a subsequentgeneration from said seed and crossing the progeny plant of a subsequentgeneration with itself or a second plant; and (c) repeating steps (a)and (b) with sufficient inbreeding until a seed of an inbred corn plantderived from the corn variety CV032539 is produced.
 18. The method ofclaim 17, further comprising crossing said inbred corn plant derivedfrom the corn variety CV032539 with a corn plant of a different genotypeto produce seed of a hybrid plant derived from the corn varietyCV032539.
 19. A method of producing a commodity plant product, themethod comprising obtaining the plant of claim 10 or a part thereof andproducing said commodity plant product therefrom.
 20. The method ofclaim 19, wherein the commodity plant product is grain, starch, seedoil, corn syrup, or protein.